CN107422006A - A kind of preparation method and application of AChE/GC HBAP/Pty/TNs optical electro-chemistry enzyme sensors - Google Patents
A kind of preparation method and application of AChE/GC HBAP/Pty/TNs optical electro-chemistry enzyme sensors Download PDFInfo
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- CN107422006A CN107422006A CN201710652025.3A CN201710652025A CN107422006A CN 107422006 A CN107422006 A CN 107422006A CN 201710652025 A CN201710652025 A CN 201710652025A CN 107422006 A CN107422006 A CN 107422006A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
Abstract
The present invention discloses a kind of preparation method and application of AChE/GC HBAP/Pty/TNs optical electro-chemistry enzyme sensors, it is characterised in that including：1) preparation of TNs electrodes；2) preparation of unimolecule GC HBAP nano particles；3) foundation of AChE/GC HBAP/Pty/TNs compound systems；The optical electro-chemistry enzyme sensor for by crosslinking agent fixation acetylcholinesterase being prepared for that there is visible light activity, and it is applied to research of the Clozapine to AChE activity influences, it was found that influence of the Clozapine to AChE activity has substrate dependence, and activation effect is shown as in the range of finite concentration.Result of study is further to study AChE activity and its provide significant experimental basis in pathogenesis, drug development, diagnosis and dosage guidance for having AChE activity changes to cause disease etc..
Description
Technical field
The present invention relates to electrochemical analysis techniques field, and it is photoelectrochemical to be specifically related to a kind of AChE/GC-HBAP/Pty/TNs
Learn the preparation method and application of enzyme sensor.
Background technology
Under the trend of aging population and infirmities of age rejuvenation, the nervus retrogression disease caused by central nervous system injury
The sick incidence of disease is constantly climbed to a higher point.The research worker for being related to the fields such as medical science, life science, chemistry has carried out such disease has been caused a disease
Many research work such as mechanism, medicament research and development and screening, treatment and prevention, wherein the research to pathogenesis turns into core
Content.The anomalous variation and central nervous system that acetylcholine esterase active is found in the research of such disease pathogenesis are damaged
Hinder that disease is closely related, acetylcholinesterase (acetylcholinesterase, AChE, EC in nerve synapse gap
3.1.1.7) major function in cholinergic system is that the transmission that neurotransmitter ACH fast hydrolyzings are made to nerve impulse terminates
Ensure cholinergic nerve system normal physiological function so as to play.When acetylcholine esterase active is suppressed, it is passed nerve
Matter ACH hydrolysis usefulness weakens, and causes a large amount of accumulations of the ACH in cholinergic nerve cynapse, flat between ACH and DA so as to cause
Weighing apparatus is destroyed.Therefore, the activity change of acetylcholinesterase is to influence the key factor of signal transduction in cholinergic nerve system.
Clinical research finds that PD and AD patient elderly patients often occur in the ill later stage with mental symptom, 25%
Early stage AD patient and more than 50% middle and advanced stage AD patient's spiritedness behavior symptom, more than 20% PD patient have hallucination.
In clinic using atypical antipsychotic drug to AD and PD etc. with Geriatric's symptom can effectively treat.Chlorine
It is hexichol phenodiazine that nitrogen, which puts down (Clozapine),Class atypical antipsychotic agents, Clozapine is used first from Seholz in 1985 etc.
Since curing PD caused by mental disorder, there is treatment successful story report successively.American Psychiatry disease association thinks that Clozapine is to control
The maximally effective medicine of Parkinsonian drugs mental disease is treated, also has the effect of obvious in control AD mental symptoms.Clozapine
Treatment PD and AD each report of dosage range differs, predominantly two classes, and one kind is that low dose of (12~20mg/ days) is long-term use of
Treatment；One kind is heavy dose of (60~65mg/ days) short-term intervention, and remission is discontinued or decrement.Although dosage differs,
Think to mental symptom good effect, shaking plasy symptom is especially trembled and Muscle tensility height is also effective.At present, Clozapine in Treating
Most effective and tolerable dosage range and the optimal course for the treatment of, there is no unified standard.
The medicine for thinking that Clozapine is commonly used as schizophreniac is had been reported that, it causes under serum cholinesterase vigor
Drop, i.e., Clozapine has the function that anticholinesterase vigor, makes DA and ACH in higher level so as to improve the concentration of acetylcholine
Establish new balance.The negative symptoms and cognition dysfunction for thinking that Clozapine can improve patient are also had been reported that simultaneously, but
Influence for AChE activity in frontal lobe does not protrude.Therefore, the mechanism that Clozapine On Cholinesterase Activity influences is still not
Clearly, it is necessary to carry out in-depth study, be pathogenesis, the inspection of the concurrent mental act diseases of nerve degenerative diseases especially AD
Survey defence, drug screening and exploitation, dosage etc. and experimental basis is provided.
At present, the most frequently used method of acetylcholine esterase active detection has following several：It is manometric method, AAS, glimmering
Light method and electrochemical methods etc..The rapid development of modern life science, medical science requires that analytical chemistry can provide more
Quickly, highly sensitive method with adapt to research needs.Optical electro-chemistry sensor make use of semiconductor nano material photoelectrochemical
To the amplification of current signal during, thus there is higher sensitivity, cause the extensive concern of research worker,
As one of study hotspot of current sensor.We utilize single polymer molecule nano particle-dioxy in research work
Change titanium nano-tube array and construct highly sensitive optical electro-chemistry sensor, and carry out Clozapine On Cholinesterase Activity shadow
Loud research.
Dissaving polymer is a kind of hyperbranchedization and has the artificial macromolecular compound of torispherical structure, its structure
Feature is containing abundant inner cavity, and carry a large amount of active function groups in end, thus it is high to show solubility, be easy into
One step carries out the chemical property of functional modification., can be by dissaving polymer based on its unique molecular structure and chemical property
Applied to the immobilization to bioactive substance.
The content of the invention
It is an object of the present invention to it is directed to the above-mentioned problems in the prior art, there is provided a kind of AChE/GC-HBAP/
The preparation method and application of Pty/TNs optical electro-chemistry enzyme sensors, GC-HBAP unimolecule nano particles are prepared for first, by it
Modify on the TNs surfaces of functionalization, fixing acetylcholinesterase by crosslinking agent is prepared for having the photoelectrochemical of visible light activity
Learn enzyme sensor, and the research influenceed applied to Clozapine On Cholinesterase Activity.
To achieve the above object, the effect above is reached, the present invention is to be achieved through the following technical solutions：
A kind of preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor, it is characterised in that including：
1) preparation of TNs electrodes
Metal titanium sheet is polishing to minute surface, is immersed in ultrasound ten minutes in acetone, ethanol, distilled water successively, then be immersed in hydrogen fluorine
Chemical polishing minute is carried out in the mixed liquor of acid and nitric acid, taking-up is rinsed, dried in the air at room temperature successively with acetone and distilled water at once
Dry, electrode middle preparation titanium sheet in homemade cylindrical electrolytic pond is negative electrode, and four graphite electrodes being symmetric are anode；
The mixed liquor of the hydrofluoric acid and nitric acid is VHF：VHNO3：VH2O=1:4:5；
2) preparation of unimolecule GC-HBAP nano particles
A synthesis gold colloid solutions (GC)：GC is synthesized with reference to Duff methods；
The synthesis of b single polymer molecule nano particles；
C unimolecule nano particles GC-HBAP synthesis；
3) foundation of AChE/GC-HBAP/Pty/TNs compound systems
Using cyclic voltammetry in TNs surfaces electropolymerization tyrasamine, three-electrode system：Working electrode is prepared TNs electricity
Pole, auxiliary electrode are platinum electrode, and reference electrode is saturated calomel electrode (SCE)；To contain 0.3mol tyrasamines and 0.04g
NaOH (0.1M) 10mL methanol solutions are electrolyte, carry out electropolymerization tyrasamine, electropolymerization current potential is 0~1.5V, and sweeping speed is
50mV/s, the circle of scanning 15, electrode is taken out, it is clean with distilled water flushing, dry, Pty/TNs modified electrodes are obtained, in electrode surface
Each μ L GC-HBAP solution of drop coating 10 in both sides, dries in 4 DEG C of refrigerators, and GC-HBAP/Pty/TNs modified electrodes are made.Finally take
In electrode side, 4 DEG C are dried 10 μ L enzyme solutions (1.0mg/mL AChE, 5.0mg/mL chitosan) drop coating, and AChE/GC- is made
HBAP/Pty/TNs optical electro-chemistry sensors.
Preferably, the preparation of step 1) the TNs electrodes, it is characterised in that：Electrolyte is：1M(NH4)2SO4In contain
0.1M NH4F and 1/12M H2C2O4, the anodic oxidation of 20V applied voltages progress 2 hours is conveyed to electrolytic cell loop.Anodic oxygen
After change terminates, titanium sheet being taken out immediately and is washed down with redistilled water, and dried up with nitrogen N 2, dried titanium sheet is transferred to horse
Not in stove, in the case where 500 DEG C of oxygen atmospheres are protected, anneal 1 hour, heating and cooldown rate are 2.5 DEG C/min.
Preferably, in the step 2) single polymer molecule nano particle synthesis, it is characterised in that：Take mol ratio every time
For 1：2.5 200mg HBAP (200mg) and PTMP (12.2mg) dissolves constant volume to 250mL with 0.8mg/mL THF, and mixing is
It is even after input catalyst 1- lauryl amines (10wt%, 1mg), be stirred at room temperature 2 hours, negate answer under vacuum condition using ethyl acetate as
Precipitating reagent is precipitated, and as in brown vacuum desiccator after precipitation is complete, is dried more than 24 hours at room temperature.
Preferably, the synthesis of step 2) the unimolecule nano particle GC-HBAP, it is characterised in that：By GC solution and list
Molecularly Imprinted Polymer nano particle is diluted with water and acetone respectively：Contain 1mg single polymer molecules in 20mL acetone solns；GC solution
By volume 1：15 dilute, and under nitrogen protective condition, the GC solution after quick agitation and dilution, polymer sickly look solution are added dropwise
Enter, the mixed solution after adding at room temperature with tri-distilled water washed by reaction overnight, test solution centrifuging and taking precipitation, repeatedly operation 3-4 times, most
After continue to employ precipitation：Unimolecule GC-HBAP nano particles.
A kind of application of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor, it is characterised in that：AChE/GC-
HBAP/Pty/TNs optical electro-chemistry enzyme sensor is applied to research of the Clozapine to AChE activity influences, finds Clozapine to AChE
The influence of activity has substrate dependence, and activation effect is shown as in the range of finite concentration.
The beneficial effects of the invention are as follows：AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor of the present invention, passes through friendship
The optical electro-chemistry enzyme sensor that acetylcholinesterase is prepared for having visible light activity is fixed in connection agent, and is applied to Clozapine pair
The research of AChE activity influences, it is found that influence of the Clozapine to AChE activity has substrate dependence, and in finite concentration scope
Inside show as activation effect.Result of study is for further research AChE activity and its in the cause for having AChE activity changes to cause disease
Anttdisease Mechanism, drug development, diagnosis and dosage guidance etc. provide significant experimental basis.
Brief description of the drawings
Fig. 1 is the preparation flow figure of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor of the present invention；
Electrode surface shape during Fig. 2 is prepared for AChE/GC-HBAP/Pty/TNs optical electro-chemistry sensor in the embodiment of the present invention
The SEM figures of looks；(A TNs, B Pty/TNs, C GC-HBAP/Pty/TNs, D AChE/GC-HBAP/Pty/TNs)
Fig. 3 is that the UV-Vis of AChE/GC-HBAP/Pty/TNs sensor preparation process in the embodiment of the present invention diffuses
Spectrogram.(curve a, b, c and d minute mark represent：TNs, Pty/TNs, GC-HBAP/Pty/TNs and AChE/GC-HBAP/Pty/TNs
The Absorption Line of modified electrode)
Embodiment
With reference to specific examples below and accompanying drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following examples.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection domain.The process of the implementation present invention,
Condition, reagent, experimental method etc., it is the universal knowledege and common knowledge of this area in addition to the following content specially referred to,
Content is not particularly limited in the present invention.
Embodiment
A kind of preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor, it is characterised in that including：
1) preparation of TNs electrodes
Metal titanium sheet is polishing to minute surface, is immersed in ultrasound ten minutes in acetone, ethanol, distilled water successively, then be immersed in hydrogen fluorine
Chemical polishing minute is carried out in the mixed liquor of acid and nitric acid, taking-up is rinsed, dried in the air at room temperature successively with acetone and distilled water at once
Dry, electrode middle preparation titanium sheet in homemade cylindrical electrolytic pond is negative electrode, and four graphite electrodes being symmetric are anode；
The mixed liquor of the hydrofluoric acid and nitric acid is VHF：VHNO3：VH2O=1:4:5；
The electrolyte is：1M(NH4)2SO4In contain 0.1M NH4F and 1/12MH2C2O4, conveyed to electrolytic cell loop
20V applied voltages carry out the anodic oxidation of 2 hours.After anodic oxidation terminates, titanium sheet is taken out immediately and is rushed with redistilled water
Only, and nitrogen N is used2Drying, dried titanium sheet is transferred in Muffle furnace, in the case where 500 DEG C of oxygen atmospheres are protected, is annealed 1 hour, is risen
Gentle cooldown rate is 2.5 DEG C/min.
2) preparation of unimolecule GC-HBAP nano particles
A synthesis gold colloid solutions (GC)：GC is synthesized with reference to Duff methods；
The synthesis of b single polymer molecule nano particles
It is 1 to take mol ratio every time：2.5 200mg HBAP (200mg) and PTMP (12.2mg) is molten with 0.8mg/mL THF
Constant volume is solved to 250mL, catalyst 1- lauryl amines (10wt%, 1mg) is put into after being well mixed, is stirred at room temperature 2 hours, negating should be true
Precipitated by precipitating reagent of ethyl acetate under empty condition, as in brown vacuum desiccator after precipitation is complete, dried at room temperature
More than 24 hours；
C unimolecule nano particles GC-HBAP synthesis
GC solution and single polymer molecule nano particle are diluted with water and acetone respectively：Contain in 20mL acetone solns
1mg single polymer molecules；GC solution by volume 1：15 dilute, under nitrogen protective condition, the GC solution after quick agitation and dilution,
Polymer sickly look solution is added dropwise, the mixed solution after adding at room temperature with three steamed by reaction overnight, test solution centrifuging and taking precipitation
Water washing, operate 3-4 times repeatedly, finally continue to employ precipitation：Unimolecule GC-HBAP nano particles.
3) foundation of AChE/GC-HBAP/Pty/TNs compound systems
Using cyclic voltammetry in TNs surfaces electropolymerization tyrasamine, three-electrode system：Working electrode is prepared TNs electricity
Pole, auxiliary electrode are platinum electrode, and reference electrode is saturated calomel electrode (SCE)；To contain 0.3mol tyrasamines and 0.04g
NaOH (0.1M) 10mL methanol solutions are electrolyte, carry out electropolymerization tyrasamine, electropolymerization current potential is 0~1.5V, and sweeping speed is
50mV/s, the circle of scanning 15, electrode is taken out, it is clean with distilled water flushing, dry, Pty/TNs modified electrodes are obtained, in electrode surface
Each μ L GC-HBAP solution of drop coating 10 in both sides, dries in 4 DEG C of refrigerators, and GC-HBAP/Pty/TNs modified electrodes are made.Finally take
In electrode side, 4 DEG C are dried 10 μ L enzyme solutions (1.0mg/mL AChE, 5.0mg/mL chitosan) drop coating, and AChE/GC- is made
HBAP/Pty/TNs optical electro-chemistry sensors.
The sign of photoelectric sensor preparation process
SEM characterizes sensor preparation process modification of surface morphology
The change of its configuration of surface can be characterized by SEM in sensor building process.Such as Fig. 2：Optical electro-chemistry senses
The SEM figures of electrode surface pattern in prepared by device；Orderly TNs (as shown in Figure 2 A), electropolymerization junket is prepared in anodic oxidation
After amine, occurs cloud form structure (as shown in Figure 2 B) in its SEM figure, because film layer is relatively thin, TNs tubular structure mays be seen indistinctly.
After modifying GC-HBAP, thicknesses of layers increase so that before the definition of TNs tubular structures is less than modification, the bright point in figure has can
Can be the GC (as shown in Figure 2 C) in GC-HBAP.AChE is fixed on to surface topography such as figure after electrode surface by chitosan
Shown in 2D, because the stop TNs tubular structures of film layer disappear.Above-mentioned phenomenon illustrates that Pty, GC-HBAP and AChE are modified successively
TNs surfaces, construct AChE/GC-HBAP/Pty/TNs sensors.
The UV-Vis DRS spectral characterization of sensor preparation process
By UV-Vis DRS spectral investigation in sensor preparation process electrode surface fluorescent property, as a result
As shown in Figure 3.As can be seen from the figure TNs modified electrodes wavelength be about 275nm and 405nm at have two strong peaks, illustrate this
Modified electrode can not only absorb ultraviolet light, additionally it is possible to absorb visible ray (curve a).After electropolymerization modification tyrasamine, Pty/TNs is repaiied
The reflectivity for adoring electrode reduces, and red shift (as shown in curve b) occurs for the absworption peak of visible region.Illustrate poly- tyrasamine to TNs's
Sensitization promotes absorption of the composite nanometer semiconductor material to visible ray.After further modifying single polymer molecule nano material
GC-HBAP/Pty/TNs reflectivity further reduces and the absworption peak of visible region slightly has red shift, illustrates single polymer molecule
GC-HBAP contributes to absorption of the composite semiconductor nano material to visible ray.Further it is also possible to it is due in GC-HBAP
AuNPs surface plasma resonances add the utilization rate to visible ray, while also illustrate that GC-HBAP is successfully modified in electrode table
Face.After AChE modifications to electrode surface, because enzyme and chitosan layer produce to light and stop and scatter, make composite to light
Absorption has declined, and reflectivity increase illustrates that enzyme molecule is successfully modified in electrode surface (as shown in curve d).It is above-mentioned existing
GC-HBAP/Pty/TNs semiconductor composite nano materials as constructed by illustrating have visible light activity, with ultraviolet section in experiment
Irradiation electrode remains to realize after limited step elimination ultraviolet light excites to semiconductor composite nano materials (GC-HBAP/Pty/TNs),
Produce light induced electron/hole pair.
Research of the Clozapine to AChE activity inhibitions
The influence to AChE activity under the horizontal Clozapine effects of 0.5mg/L, 1mg/L two is studied, because blood accounts for people
The 7.5-8% of body body weight, density is between 1.05-1.06g/mL, and blood flow volume is about 4.5L in the adult body of 60kg body weight, the above
Concentration of Clozapine is respectively equivalent to dosage in clinical treatment：6.75mg/d(day)、13.5mg/d.
For convenience of the change of AChE activity in Photoelectric Detection is discussed, relative activity IR is defined：
IR=IATCh/I0× 100%
I in formulaATCh、I0The increasing of photoelectric current when indicating respectively, acting on lower addition finite concentration substrate A TCh without Clozapine
Amount.
The horizontal lower AChE relative activity changes of 1 different concentration of Clozapine of table
It is to the shadow of AChE activity under two levels of 0.5mg/L, 1mg/L to have investigated concentration of Clozapine by experiment in vitro
Ring, research is found, influence of the Clozapine to AChE activity embodies the feature of concentration of substrate dependence, as shown in table 1, when substrate is dense
(0.05mM) I when spending relatively lowR<100%, illustrate that its influence to AChE activity shows as suppressing.After concentration of substrate increases 10 times,
Relative activity under two concentration of Clozapine are horizontal is all higher than 100%, shows as activating.With the increase of concentration, relative activity
Start to reduce again.It is also observed that when work occurs during Clozapine is carried out to enzyme injury experiment in same branch sensor
Sexual abnormality increases, situation about then reducing suddenly.Speculate, it may be possible to which configuration change occurs under the mechanism activated repeatedly in AChE
Enzyme is caused to inactivate.This is likely to be Clozapine in clinic and is used in auxiliary treatment AD and PD patient's late period concurrent mental symptom to AD
One of with the mechanism of PD symptoms.Experimental result explain to a certain extent in clinic using high-dose therapy method in short-term and it is long when it is low
Measurement Law carries out the reason for auxiliary treatment.In addition speculate also be probably Clozapine metabolite to AChE activity produce suppress institute
Cause.Result of study is further research AChE activity and its is having AChE activity changes to cause the pathogenesis of disease, medicine to be opened
Hair, diagnosis and dosage guidance etc. provide significant experimental basis.
The foregoing description of the disclosed embodiments, professional and technical personnel in the field are enable to realize or using the present invention.
A variety of modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, it is of the invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The most wide scope caused.
Claims (6)
- A kind of 1. preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor, it is characterised in that including：1) preparation of TNs electrodesMetal titanium sheet is polishing to minute surface, is immersed in acetone, ethanol, distilled water ultrasound successively ten minutes, then be immersed in hydrofluoric acid and Carried out in the mixed liquor of nitric acid chemical polishing minute, taking-up is rinsed successively with acetone and distilled water at once, is dried at room temperature, electricity Pole middle preparation titanium sheet in homemade cylindrical electrolytic pond is negative electrode, and four graphite electrodes being symmetric are anode；2) preparation of unimolecule GC-HBAP nano particlesA synthesis gold colloid solutions (GC)：GC is synthesized with reference to Duff methods；The synthesis of b single polymer molecule nano particles；C unimolecule nano particles GC-HBAP synthesis；3) foundation of AChE/GC-HBAP/Pty/TNs compound systemsUsing cyclic voltammetry in TNs surfaces electropolymerization tyrasamine, three-electrode system：Working electrode is prepared TNs electrodes, auxiliary It is platinum electrode to help electrode, and reference electrode is saturated calomel electrode (SCE)；To contain 0.3mol tyrasamines and 0.04g NaOH The 10mL methanol solutions of (0.1M) are electrolyte, carry out electropolymerization tyrasamine, and electropolymerization current potential is 0~1.5V, and it is 50mV/s to sweep speed, The circle of scanning 15, electrode is taken out, it is clean with distilled water flushing, dry, obtain Pty/TNs modified electrodes, it is each in electrode surface both sides The μ L GC-HBAP solution of drop coating 10, dries in 4 DEG C of refrigerators, and GC-HBAP/Pty/TNs modified electrodes are made, finally take 10 μ L enzymes In electrode side, 4 DEG C are dried solution (1.0mg/mL AChE, 5.0mg/mL chitosan) drop coating, and AChE/GC-HBAP/ is made Pty/TNs optical electro-chemistry sensors.
- 2. the preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor as claimed in claim 1, its feature It is：The mixed liquor of the preparation of step 1) the TNs electrodes, hydrofluoric acid and nitric acid is VHF：VHNO3：VH2O=1:4:5.
- 3. the preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor as claimed in claim 1, its feature It is：The preparation of step 1) the TNs electrodes, electrolyte are：1M(NH4)2SO4In contain 0.1M NH4F and 1/12M H2C2O4, The anodic oxidation of 2 hours is carried out to electrolytic cell loop conveying 20V applied voltages, after anodic oxidation terminates, takes out titanium sheet immediately And washed down with redistilled water, and use nitrogen N2Drying, dried titanium sheet is transferred in Muffle furnace, is protected in 500 DEG C of oxygen atmospheres Under shield, anneal 1 hour, heating and cooldown rate are 2.5 DEG C/min.
- 4. the preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor as claimed in claim 1, its feature It is：The synthesis of single polymer molecule nano particle in the step 2), it is 1 to take mol ratio every time：2.5 200mg HBAP (200mg) dissolves constant volume to 250mL with PTMP (12.2mg) with 0.8mg/mL THF, and catalyst 1- ten is put into after being well mixed Diamines (10wt%, 1mg), it is stirred at room temperature 2 hours, negates to answer and precipitated under vacuum condition by precipitating reagent of ethyl acetate, sinks As in brown vacuum desiccator after forming sediment completely, dry more than 24 hours at room temperature.
- 5. the preparation method of AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor as claimed in claim 1, its feature It is：The synthesis of step 2) the unimolecule nano particle GC-HBAP, GC solution and single polymer molecule nano particle are distinguished Diluted with water and acetone：Contain 1mg single polymer molecules in 20mL acetone solns；GC solution by volume 1：15 dilutions, nitrogen Under protective condition, the GC solution after quick agitation and dilution, polymer sickly look solution is added dropwise, the mixed solution room after adding The lower reaction overnight of temperature, test solution centrifuging and taking precipitation are washed with tri-distilled water, operate 3-4 times repeatedly, finally continue to employ precipitation：Unimolecule GC- HBAP nano particles.
- 6. the application of the AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensors as prepared by claim 1-5, its feature It is：AChE/GC-HBAP/Pty/TNs optical electro-chemistry enzyme sensor is applied to research of the Clozapine to AChE activity influences, hair Influence of the existing Clozapine to AChE activity has substrate dependence, and activation effect is shown as in the range of finite concentration.
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| CN110161092A (en) * | 2019-03-28 | 2019-08-23 | 玉溪师范学院 | A kind of AchE/GQDs/N-F-TiO2Ns Photoelectrochemistrbiosensor biosensor preparation method and applications |
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